The hyperthermophilic archaea are microorganisms that grow optimally at a temperature above 80° C. Many species of these extremely thermophilic bacteria-like organisms have been isolated, mainly from volcanically and geothermally heated hydrothermal environments, such as solfataric fields, hot springs, and submarine hot vents.
The discovery of microorganisms growing optimally around 80° C. is of considerable interest in both academic and industrial communities. Both the organisms and their enzymes have the potential to bridge the gap between biochemical catalysis and many industrial chemical conversions. However, knowledge of the metabolism of the hyperthermophilic microorganisms is presently very limited.
In many hyperthermophilic archaea habited in these biotops, the order Sulfolobales which includes the genus Sulfolobus, have a chemolithoautotrophic metabolism which converts elemental sulfur to hydrogen sulfide using organic compounds or hydrogen as an electron donor. Although Sulfolobus is the sulfur-oxidizing genus, this genus can grow chemoheterotrophically to a high cell density using sugars. Sulfolobus solfataricus optimally grows at 80-85° C. and pH 2-4, utilizing glucose as the sole carbon and energy source (Grogan, J. Bacteriol. 171:6710-6719, 1989)). In Sulfolobus, the glucose metabolism pathway was first analyzed with 14C-glucose-label experiments by De Rosa et al. (Biochem. J. 224: 407-414, 1984). De Rosa's experiment shows that Sulfolobus can convert glucose to pyruvate through a modified Entner-Doudoroff (ED) pathway which produces non-phosphorylated intermediates such as gluconate, 2-keto-3-deoxygluconate (KDG), and glyceraldehyde. The first reaction of the non-phosphorylated ED pathway in S. solfataricus involves the NAD(P)+-dependent oxidation of glucose to gluconate, catalyzed by glucose dehydrogenase. Gluconate is then dehydrated by gluconate dehydratase (EC 4.2.1.39) to 2-keto-3-deoxygluconate (KDG), which is cleaved to pyruvate and glyceraldehydes, and catalyzed by KDG-alolase (EC 4.1.2.20). The modified ED pathway involving non-phosphorylated intermediates was also discovered in thermoacidophilic archaeon Thermoplasma acidophilum (Budgen et al. FEBS Lett. 196:207-210, 1986). The Thermoplasma acidophilum metabolizes glyceraldehyde formed via this non-phosphorylated route by glyceraldehyde dehydrogenase to glycerate, which is phosphorylated to form 2-phosphoglycerate. This intermediate is then converted to generate one molecule of pyruvate by enolase and pyruvate kinase. The non-phosphorylated ED pathway is a unique glycolysis pathway discovered only in the thermoacidophilic archaea, S. solfataricus and T. acidophilum. FIG. 1 is a non-phosphorylated ED pathway.
Another modified ED pathway involving phosphorylated intermediates is known as a novel glycolysis route for glucose conversion to pyruvate in some species. This metabolism was first discovered by Szymona et al. from eubacteria Rhodobacter sphaeroides, and was also later found from Clostridia sp. and halobacteria (Conway, FEMS Microbiol. Rev. 103:1-28, 1992). In this pathway, KDG produced by gluconate dehydratase is phosphorylated by KDG kinase to 2-keto-3-deoxy-6-phosphogluconate (KDPG) and is then cleaved by KDPG aldolase to pyruvate and glyceraldehyde-3-phosphate. The latter intermediate is oxidized to pyruvate, a process that involves a conventional route, via glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase, enolase, and pyruvate kinase.
Gluconate dehydratase has described by Kersters et al., Antonie van Leeuwenhoek. 37: 233-246 (1971); Kersters et al., Methods Enzymol. 42: 301-304 (1975); Bender et al., Eur. J. Biochem. 40: 309-321 (1973); Bender et al., Methods Enzymol. 90: 283-287 (1982). The protein was purified and characterized only from bacteria, Achromobacter species, and Clostridium pasteurianum, which metabolize gluconate via a former glycolysis pathway. A comparison of the biochemical properties of each enzymes shows that they are very different despite in vivo the same catalytic reaction. In thermoacidophilic archaea, S. solfataricus, and T. acidophilum, however biochemical properties and detail mechanisms of the gluconate dehydratases are still unknown. Despite characterizations of two enzymes from the above-described bacteria, no genes encoding gluconate dehydratase or partial amino acid sequences have been reported. Hence, although recently the genomes of S. solfataricus and T. acidophilum were completely sequenced, putative genes encoding gluconate dehydratase could not be annotated in the database (She et al., Proc. Natl. Acad. Sci. USA. 98: 7835-7840, 2001; Ruepp et al., Nature 407:508-513, 2000). In addition, the known gluconate dehydratases do not maintain thermostability at temperatures greater than about 50° C. for prolonged periods up to several hours. Thus it is necessary to develop a novel gluconate dehydratase that can retain activity at high temperatures for prolonged periods of time.